RESUMO
Perinatal asphyxia (PA) is one of the most frequent risk factors for several neurodevelopmental disorders (NDDs) of presumed multifactorial etiology. Dysfunction of neuronal connectivity is thought to play a central role in the pathophysiology of NDDs. Because underlying causes of some NDDs begin before/during birth, we asked whether this clinical condition might affect accurate establishment of neural circuits in the hippocampus as a consequence of disturbed brain plasticity. We used a murine model that mimics the pathophysiological processes of perinatal asphyxia. Histological analyses of neurons (NeuN), dendrites (MAP-2), neurofilaments (NF-M/Hp) and correlative electron microscopy studies of dendritic spines were performed in Stratum radiatum of the hippocampal CA1 area after postnatal ontogenesis. Protein and mRNA analyses were achieved by Western blot and RT-qPCR. Behavioral tests were also carried out. NeuN abnormal staining and spine density were increased. RT-qPCR assays revealed a ß-actin mRNA over-expression, while Western blot analysis showed higher ß-actin protein levels in synaptosomal fractions in experimental group. M6a expression, protein involved in filopodium formation and synaptogenesis, was also increased. Furthermore, we found that PI3K/Akt/GSK3 pathway signaling, which is involved in synaptogenesis, was activated. Moreover, asphyctic animals showed habituation memory changes in the open field test. Our results suggest that abnormal synaptogenesis induced by PA as a consequence of excessive brain plasticity during brain development may contribute to the etiology of the NDDs. Consequences of this altered synaptic maturation can underlie some of the later behavioral deficits observed in NDDs.
Assuntos
Asfixia/patologia , Hipocampo/fisiopatologia , Plasticidade Neuronal/fisiologia , Análise de Variância , Animais , Asfixia/fisiopatologia , Aprendizagem da Esquiva/fisiologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Células Piramidais/metabolismo , Células Piramidais/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.
Assuntos
Humanos , Animais , Actinas/metabolismo , Amarelo de Eosina-(YS)/farmacologia , Amarelo de Eosina-(YS)/metabolismo , Foto-Oxidação , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Coloração e Rotulagem/métodos , Corantes Fluorescentes/farmacologia , Faloidina/farmacologia , Imageamento Tridimensional/métodos , Modelos Moleculares , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Microscopia de Fluorescência/métodos , Oxirredução , FótonsRESUMO
Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.(AU)